TWI679985B - Tie2 activator with olive fruit extract - Google Patents

Abstract

The object of the present invention is to provide a Tie2 activator or the like that exerts a vascular permeability inhibitory effect by stabilizing vascular endothelial cells by inducing adhesion between vascular endothelial cells.

The solution is to provide Tie2 activator and the like by containing olive fruit extract as an active ingredient.

Description

Tie2 activator with olive fruit extract

The present invention relates to a Tie2 activator, and more specifically to a Tie2 activator containing an olive fruit extract as an active ingredient. The present invention also relates to vascular permeability inhibitors, angiogenesis inhibitors, vascular maturation agents, vascular normalizers, vascular stabilizers, and lymphatic vessel stabilizers containing olive fruit extract as active ingredients. Agent. The present invention also relates to a vascular permeability inhibitor, a vascular maturation agent, and a blood vessel that contain one or more components selected from the group consisting of tyritol hydrate, tyrosol, and oleuropein as an active ingredient. Normalizer, vascular stabilizer, lymphatic stabilizer, and Tie2 activator. Furthermore, this invention relates to the composition containing the said agent.

Blood vessels not only play the role of transmitting oxygen or nutrients to the organs of the body through the blood, but also the role of transporting metabolic waste from tissues to the body and excreting them to the outside of the body. They are extremely important tissues in the human body in maintaining life activities. Generally speaking, due to the aging of blood vessels or functional failure, as represented by arteriosclerosis, it will seriously affect the health of the human body. Therefore, it is also closely related to external symptoms such as wrinkles or edema. In recent years, against this blood Tube aging and the activation of the receptor-type tyrosine kinase Tie2 (Tyrosine kinase with Ig and ECF homology domain 2) have attracted much attention. When Tie2 is activated by angiogenin from the wall cells of blood vessels, adhesion between vascular endothelial cells can be induced, which is known to contribute to the stabilization of vascular endothelial cells. In recent years, a large number of ingredients or foods with Tie2 activation have been developed to supplement foods or beverages, and to be used for the purpose of beauty or improving edema and cold hands and feet.

Specifically, this Tie2 activation can be used in soap bark, yellow wolfberry, ginkgo, oyster, turmeric, chrysanthemum, jujube, wolfberry, chamomile, gorse, hawthorn, star fruit, moon peach, lotus, South African red bush tea, capers, Chinese pear, guava, ravioli, acanthopanax senticosus, mango ginger, ginseng, milk seed, wingless pigweed, spinosa, loosestrife, hemerocallis, wild wild taro, province oil, Changzhou, Haizhou, Qiliteng, Xiaochiche Its effect was found in the extracts of P. angustifolia, Quercus acutissima, Quercus acutissima, Star apple, Psyllium chinensis, Prunus mume, Bayberry, Cinnamon, Saponaria, Polygonatum, Polygonatum odoratum, Guarulene, Morinda officinalis, etc. (patent References 1 to 7).

On the other hand, as ingredients, ursolic acid, corosolic acid, 3-O-galloproanthocyanidin B-1, linolenic acid, 13-hydroxy-9Z, 11E, 15E-octadecadiene Acid, proanthocyanidin B-2, epicatechin- (4β-6) -epicatechin (4β-8) -epicatechin, proanthocyanidin C-1, baicalin VIII, soybean Saponin I, 3'-O-methyl gallocatechin, Pipeernonaline, syringoresinol, Eleutheroside E, Eleutheroside E1, sesamin, Eudesmine, Sylvatesmin, pinoresinol, Yangambin, Forsythinol, coumarin, etc. Effective (Patent Documents 8 to 10).

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 2012-236795

[Patent Document 2] Japanese Patent Publication No. 2009-154237

[Patent Document 3] Japanese Patent Laid-Open No. 2011-201811

[Patent Document 4] Japanese Patent Laid-Open No. 2011-102275

[Patent Document 5] Japanese Patent Application Laid-Open No. 2011-102274

[Patent Document 6] Japanese Patent Laid-Open No. 2011-102273

[Patent Document 7] Japanese Patent Laid-Open No. 2009-263358

[Patent Document 8] Japanese Patent Laid-Open No. 2014-97977

[Patent Document 9] Japanese Patent Laid-Open No. 2013-241356

[Patent Document 10] Japanese Patent Application Laid-Open No. 2011-102272

The object of the present invention is to provide a novel component having excellent Tie2 activation and the like, which can stabilize vascular endothelial cells and exert vascular permeation inhibitory effects by inducing adhesion between vascular endothelial cells.

In order to solve the above-mentioned problems, as a result of intensive research conducted by the inventors of the present case, it has been discovered that components obtained from olives, and more preferably olive fruits, have Tie2 activation and the like. It has also been found that tyramine hydrate, tyrosol, and oleuropein have vascular permeability inhibitory effects, vascular maturation effects, and positive vascular effects. The normalization, the stabilization of blood vessels, the stabilization of lymphatic vessels, and the activation of Tie2 finally complete the present invention.

That is, aspects of the present invention include the following inventions, but are not limited thereto.

(1) A Tie2 activator containing an olive fruit extract as an active ingredient.

(2) The Tie2 activator according to (1), wherein the olive fruit extract is obtained from olive juice.

(3) The Tie2 activator according to (1) or (2), wherein the olive fruit extract is soluble in an aqueous solvent.

(4) The Tie2 activator according to any one of (1) to (3), wherein the olive fruit extract system contains a compound selected from polyphenols, compounds containing these in the structure, and terpene moieties derived from these compounds One or more components of the group of cleavage ether terpenes.

(5) The Tie2 activator according to any one of (1) to (4), wherein the olive fruit extract system contains at least one selected from the group consisting of tyranol hydrate, tyrosol, and oleuropein Of ingredients.

(6) A composition containing the Tie2 activator according to any one of (1) to (5).

(7) The composition as described in (6), which is marked with a function exhibited by activating Tie2.

(8) A vascular permeability inhibitor comprising an olive fruit extract as an active ingredient.

(9) A vascular maturation agent containing olive fruit extract Substance as an active ingredient.

(10) A blood vessel normalizing agent containing an olive fruit extract as an active ingredient.

(11) A blood vessel stabilizer containing an olive fruit extract as an active ingredient.

(12) A lymphatic vessel stabilizer containing an olive fruit extract as an active ingredient.

(13) A composition containing the medicament according to any one of (8) to (12).

(14) The composition according to (13), which is added with a function selected from the function exerted by vascular permeability inhibition, the function exerted by aging of blood vessels, the function exerted by normalization of blood vessels, Indication of one or more functions in the group formed by the function of the blood vessel stabilization and the function of the lymphatic tube stabilization.

(15) A vascular permeability inhibitor comprising, as an active ingredient, one or more components selected from the group consisting of tyritol hydrate, tyrosol, and oleuropein.

(16) A vascular maturation agent containing, as an active ingredient, one or more components selected from the group consisting of tyritol hydrate, tyrosol, and oleuropein.

(17) A blood vessel normalizing agent containing, as an active ingredient, one or more components selected from the group consisting of tyritol hydrate, tyrosol, and oleuropein.

(18) A blood vessel stabilizing agent, which contains a substance selected from the group consisting of hydrated casein One or more components in a group of amine alcohol, tyrosol, and oleuropein are used as effective components.

(19) A lymphatic vessel stabilizing agent containing, as an active ingredient, one or more components selected from the group consisting of tyramine hydrate, tyrosol, and oleuropein.

(20) A Tie2 activator containing, as an active ingredient, one or more components selected from the group consisting of tyritol hydrate, tyrosol, and oleuropein.

(21) A composition containing the medicament according to any one of (15) to (20).

(22) The composition according to (21), which is added with a function selected from the function exerted by suppression of vascular permeability, the function exerted by maturation of blood vessels, the function exerted by normalization of blood vessels, Indication of one or more functions in a group of functions exhibited by stabilization of blood vessels, a function of stabilization of lymphatic vessels, and functions exhibited by activation of Tie2.

The Tie2 activator of the present invention, by virtue of the excellent Tie2 activation of olive fruit extract, induces adhesion between vascular endothelial cells, stabilizes vascular endothelial cells, and inhibits vascular infiltration, thereby improving symptoms caused by aging of blood vessels or lymphatic vessels. And prevent it. For example, through the adhesion induction of vascular endothelial cells produced by Tie2 activation, the cells of blood vessels are tightly adhered, and nutrients are spread throughout the skin to obtain skin tension and texture. Beauty effects such as improvement, prevention of wrinkles. In addition, by filling the space between the endothelial cells of blood vessels and lymphatic vessels, the blood circulation can be improved, and water from the tissues can be recovered. Metabolic waste can be improved, preventing cold hands and feet, stiff shoulders, edema, dark circles, etc. Effect of symptoms.

In addition, the medicament containing the olive fruit extract of the present invention as an active ingredient can exert vascular permeability inhibition, vascular maturation, normalization of blood vessels, stabilization of blood vessels, and stabilization of lymphatic vessels. Furthermore, the medicament of the present invention containing one or more components selected from the group consisting of tyritol hydrate, tyrosol, and oleuropein can also exert vascular permeability inhibitory effect, vascular maturation effect, Normalization of blood vessels, stabilization of blood vessels, stabilization of lymphatic vessels, and activation of Tie2.

FIG. 1 is a graph showing the results of the Western blotting method of the activated Tie2 of each sample and the Tie2 activation rate compared with the control group.

FIG. 2 is a graph showing the results of the Myers analysis method and the vascular permeability inhibition rate of each sample.

Hereinafter, embodiments of the present invention will be described in detail.

Tie2 activator

Capillary structural stabilization Activation of a receptor called Tie2 plays an important role. This Tie2 shows that in addition to vascular endothelial cells, it also exists in lymphatic endothelial cells. Activation by angiogenin released by parietal cells can enhance the adhesion of endothelial cells to each other or parietal cells, and stabilize the vascular structure. related. The "Tie2 activator" in the present invention refers to an agent having the ability to convert Tie2 into its activator (phosphorylated Tie2) by phosphorylation. The Tie2 activator of the present invention is a Tie2 activating agent Olive fruit extracts include, as an active ingredient, polyphenols such as tyritol and tyrosol, as well as oleuropein, Orasein, eugenol, and 3,4-DHPEA in the structure. -EA, 3,4-DHPEA-EDA, hydrated tyrosin glycoside, 4-Ep-coumaroylsecologanoside with p-coumaric acid in the structure. Furthermore, most of these polyphenol derivatives have compounds derived from terpenes in their molecules, and their terpenes are cleavenene ether terpenes. Examples of other components include hydrated tyrosine, rengiosides and oleosides, which are terpene glycosides. The active ingredient in the Tie2 activator of the present invention is not particularly limited, but is preferably selected from the group consisting of hydrated tyranol, tyrosol, oleuropein, Orasein, syringin, 3,4-DHPEA-EA, and 3,4 One or more components in the group formed by -DHPEA-EDA are more preferably one or more components selected from the group formed by hydrated tyritol, tyrosol, and oleuropein.

Others have suggested that the activity of this Tie2, for example, will decrease with age; it can be seen that blood vessels or lymphatic vessels will gradually age with this. Therefore, the Tie2 activator of the present invention can prevent and improve the aging of blood vessels or lymphatic vessels through the excellent Tie2 activation of olive fruit extract.

In addition, if the vascular structure becomes unstable due to aging of the blood vessels, nutritional components such as plasma components will leak out of the blood vessels, making it impossible to transport the required components to the required site. As a result, it is thought that various problems such as deterioration of the skin state may occur. Therefore, the Tie2 activator of the present invention can stabilize vascular endothelial cells and exert vascular permeability inhibition by virtue of the excellent Tie2 activation of olive fruit extract. In addition, the cells of blood vessels are tightly adhered to allow nutrition to permeate the skin, which can achieve cosmetic effects such as improvement of skin tension and texture, and prevention of wrinkles. In addition, by filling the space between endothelial cells of blood vessels and lymphatic vessels, blood circulation can be improved, and the recovery of water and metabolic waste from tissues can improve and prevent cold hands and feet, stiff shoulders, edema, dark circles, etc. The effect of each symptom.

<Active ingredient (olive fruit extract)>

In the present invention, "olive fruit extract" refers to an extract obtained from olive fruit extraction, and refers to having Tie2 activity, vascular permeability inhibition, vascular maturation, vascular normalization, and vascular Stabilizing and lymphatic stabilizing components. More preferably, it is an ingredient group obtained from olive juice. As used herein, the term “olive juice” refers to an extract obtained from an aqueous layer obtained by separating a liquid phase obtained from an olive fruit into an oil layer and an aqueous layer. The olive fruit extract is, for example, a water-soluble component, and can also be extracted from olives using an aqueous solvent. Examples of the aqueous solvent include water and alcohols (for example, ethanol). Olive fruit extracts can be used directly as olive juice or aqueous solvent extracts. After filtering these substances, the polyphenol was concentrated by an adsorption column and spray-dried.

The content of olive fruit extract contained in the Tie2 activator is 0.01% to 50% by weight, preferably 0.1% to 40% by weight, and more preferably 1% to 30% by weight relative to the total amount of the Tie2 activator. weight%. The dosage of the Tie2 activator of the present invention can be appropriately determined according to the content of the olive fruit extract contained in the Tie2 activator, and can also be appropriately determined based on the age, weight, health status, etc. of the subject to be used, for example, one day for a human adult The ingestion amount is 0.1 mg to 400 mg, preferably 1 mg to 200 mg, and more preferably 10 mg to 100 mg. It can be ingested and administered in single or divided doses.

As a component of the olive fruit extract of the present invention, in addition to polyphenols, it also contains one selected from the group consisting of cleavenene ether terpenes derived from the compounds containing these compounds or the terpene moiety of these compounds. The above ingredients. Examples of polyphenols include polyphenols such as hydrated tyritol and tyrosol, and oleuropein, Orasein, syringin, 3,4-DHPEA-EA, 3 containing them in the structure. , 4-DHPEA-EDA, hydrated tyrosin glycoside, 4-Ep-coumaroylsecologanoside with p-coumaric acid in the structure. Furthermore, most of these polyphenol derivatives have compounds derived from terpenes in their molecules, and their terpenes are cleavenene ether terpenes. Examples of other components include hydrated tyrosine, rengiosides and oleosides, which are terpene glycosides. The active ingredient in the present invention is not particularly limited, but is preferably selected from the group consisting of hydrated tyritol, tyrosol, oleuropein, Orasein, syringin, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA One or more components in the formed group, especially selected from the group consisting of tyramine hydrate, tyrosol, and oleuropein One or more components in the group are more preferable.

<Raw material of olive fruit extract>

Olives used as raw materials for olive fruit extracts are plants of the genus Asteraceae, and olive oil derived from the fruits of olives is widely used as edible oil. On the other hand, the fruit extract of olives is one of the industrial wastes generated when olives are pressed into oil. In recent years, people have developed or sold materials for health foods or supplements processed from them. As the main raw material of the olive fruit extract, olive fruit can be used, but leaves, seeds, leaves, stems, etc. may be mixed therein. When olive fruit is used as a raw material, it can be used in a raw state, or it can be used after being dried by freeze-drying or the like. Moreover, the residue after squeezing oil from an olive fruit may be used as it is, or it may be used in a dry state. Preferably, only the aqueous layer obtained by separating the liquid phase obtained from the olive fruit into an oil layer and an aqueous layer, that is, olive juice, is used as a raw material.

The varieties of olives are not particularly limited, and for example, varieties such as Manzanillo, Lucca, Nevadillo blanco, Mission, Picual, Arbechina, Hojiblanca, Cornicabra, Gordal, Moraiolo, Frantoio, Coratina, Leccino and the like can be applied.

<Method for preparing olive fruit extract>

The olive fruit extract can be obtained through a separation step and a purification step.

(Separation step)

Press the olive fruit as raw material and separate it into oil by centrifugation With fruit juice, get olive juice. Pressing and centrifuging can be performed by methods known to those skilled in the art.

(Refining step)

The extract obtained by filtering and concentrating the olive juice obtained in the separation step can also be used as the olive fruit extract of the present invention. In addition, by filtering a filtrate obtained by filtering olive juice into a column filled with an adsorption resin, components such as sugars, minerals, and organic acids that are not adsorbed on the adsorption resin are removed, and then an aqueous solvent such as water, Ethanol, or a mixture of these) is eluted to obtain a dissolution fraction that is rich in components having Tie2 activation from olives and the like. It is also possible to use this eluted fraction rich in components having Tie2 activation and the like as the olive fruit extract of the present invention. Those skilled in the art can appropriately set the conditions of the type of adsorption resin or the dissolution solvent of the column according to the type and shape of the olive used.

Other potions

As another aspect of the present invention, vascular permeability inhibitors, angiogenesis inhibitors, vascular mature agents, vascular normalizers, vascular stabilizers, and lymphatic vessel stabilizers can be mentioned. These medicines contain olive fruit extract, which is also an active ingredient of the above-mentioned Tie2 activator, as an active ingredient. In addition, these agents may be selected from the group consisting of tyranol hydrate, tyrosol, oleuropein, Orasein, syringin, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA. 1 or more ingredients as effective ingredients It is more preferable to contain, as an active ingredient, one or more components selected from the group consisting of tyranol hydrate, tyrosol, and oleuropein.

In the present invention, the "vascular permeability inhibitor" refers to an agent having an inhibitory effect on an increase in vascular permeability caused by VEGF (vascular endothelial cell growth factor). "Angiogenesis inhibitor" refers to an agent having the effect of inhibiting a new blood vessel network formed by an existing blood vessel. The "vascular maturation agent" refers to an agent that induces adhesion between vascular endothelial cells and vascular wall cells and forms a maturation effect that prevents the uneven adhesion of vascular endothelial cells between environmental factors in blood vessels that can easily leak out of blood vessels. "Vascularizing agent" refers to a blood vessel endothelial cell that enhances adhesion of vascular endothelial cells to each other, and promotes the strengthening of vascular endothelial cells by vascular wall cells, thereby causing abnormal blood vessels such as vascular permeability failure or abnormal blood vessels with disordered vascular growth. Medicament for normalizing normal conditions. "Vascular stabilizer" means that it has the effect of inhibiting the damage to existing blood vessels, detachment of vascular endothelial cells from each other, detachment of vascular endothelial cells and vascular wall cells, and stabilization of cell death of vascular endothelial cells. Functioning medicament. "Lymphatic vessel stabilizing agent" refers to an agent that has the function of stabilizing the lymphatic vessels, appropriately disposing and reinforcing the endothelial cells of the lymphatic vessels, and thereby maintaining the rapid recovery function of interstitial fluid.

Vascular permeability inhibitors, angiogenesis inhibitors, vascular ripening agents, vascular normalizing agents, vascular stabilizing agents, and lymphatic stabilizing agents can be directly applied to the types, amounts, and manufacturing methods of the components used Tie2 activator is about the content described.

Composition

As one aspect of the present invention, there may be selected from the group consisting of the Tie2 activator, vascular permeability inhibitor, angiogenesis inhibitor, vascular maturation agent, vascular normalization agent, vascular stabilizing agent, and lymphatic vessel stabilization. A composition of one or more medicaments in a group of chemical agents.

In addition to the Tie2 activator, the composition may be mixed with well-known additives used in cosmetics, pharmaceuticals, and the like. The additives are not particularly limited, and additives such as excipients, binding agents, disintegrating adjuvants, lubricants, antioxidants, preservatives, flavoring agents, flavoring agents, colorants, and flavors can be used. For example, L-ascorbic acid, catechin, dextrin, cyclodextrin, calcium carbonate, trehalose, and the like can be used. In addition, the composition may be used in combination with a well-known component having Tie2 activating effect or the like, and may be used in combination with a component known to be effective in preventing aging of blood vessels or being cosmetic. For example, soap bark, wolfberry, ginkgo, ginkgo, oyster, turmeric, chrysanthemum, jujube, wolfberry, chamomile, gorse, hawthorn, star fruit, moon peach, lotus, South African red shrub tea, capers, which are reported to have Tie2 activation , Rosewood, guava, ravioli, acanthopanax senticosus, mango ginger, ginseng, milk seed, wingless pig's hairy cabbage, spinosa, loosestrife, hemerocallis, wild wild taro, province oil, Changzhou, Haizhou, Qiliteng, Xiaochi Extracts from Che, Quercus acutissima, Quercus acutissima, Angustifolia, Star Apple, Psyllium, Scutellaria, Bayberry, Cinnamon, Saponin, Polygonatum sibiricum, Polygonatum odoratum, Guarulene, Morinda officinalis or contain extracts that improve vascular endothelium It is an effective proanthocyanidin extract of grape seeds or pine bark, peanut seed coat, litchi seed coat, etc., or amino acids such as L-citrulline, L-arginine.

The form of the composition can be appropriately determined depending on the purpose of use, and can be used in the form of a cosmetic composition, a pharmaceutical composition, or the like. Pharmaceutical composition, except pharmaceuticals and In addition to quasi-pharmaceuticals, although they are not included in the Pharmaceutical Affairs Law, they also contain the same components as pharmaceuticals or quasi-pharmaceuticals which are expected to have the preventive and improving effects of the Tie2 activator of the present invention, Or it expressly or implicitly asks for a composition for preventing and improving the effect produced by the Tie2 activator and the like of the present invention. The dosage form of the composition is also not particularly limited, and is typically an oral preparation such as a granule, a lozenge, a capsule, a liquid, etc., preferably a capsule.

Another aspect of the present invention relates to a label containing a function exerted by inhibition of vascular permeability, maturation of blood vessels, normalization of blood vessels, stabilization of blood vessels, stabilization of lymphatic vessels, or activation of Tie2. Composition of the medicament of the present invention. Such a label or function label is not particularly limited, and examples thereof include "inhibition of vascular hyperpermeability", "inhibition of leakage of intravascular factors to the outside of blood vessels", "improvement of blood vessels to a normal state", and "maintenance of blood vessels in a normal state" State "," inhibition of vascular endothelial cells from each other "," inhibition of cell death of vascular endothelial cells ", or" maintaining rapid recovery of interstitial fluid "and the like. In the present invention, such a label and a function label may be added to the composition itself, or may be added to a container or a package of the composition.

The composition system according to one aspect of the present invention contains 0.01% to 99% by weight, preferably 0.1% to 50% by weight, and more preferably 1% to 30% by weight relative to the total amount of the composition. More preferably, it is 10% to 30% by weight of Tie2 activator, and contains 0.05% to 50% by weight, more preferably 0.5% to 25% by weight, and even more preferably 5% by weight relative to the total composition. ~ 20% by weight olive fruit extract. The amount of the composition according to one aspect of the present invention varies with the content of the olive fruit extract contained in the Tie2 activator, and can be appropriately determined based on the age, weight, health status, etc. of the subject to be used. The daily intake is 0.1mg ~ 400mg, preferably 1mg ~ 300mg, more preferably 10mg ~ 200mg. It can be ingested and administered in single or divided multiple times.

Moreover, as one aspect of the present invention, those containing the vascular permeability inhibitor, angiogenesis inhibitor, vascular maturation agent, vascular normalization agent, vascular stabilizing agent, or lymphatic stabilizing agent are mentioned.组合 物。 Composition. The types, forms, dosage forms, dosages, and the like of the components of these compositions can be directly applied to the contents described in relation to the composition containing the Tie2 activator.

[Example]

Hereinafter, the present invention will be described more specifically based on examples. Furthermore, the present invention is not limited by these examples.

Example 1: Evaluation of Tie2 activation of olive fruit extract

Olive fruit extract was used to assess Tie2 activation.

<Preparation of sample (olive fruit extract)>

The olive fruit is pressed and centrifuged to separate the oil and juice. Here, after filtering the obtained juice, the components of the adsorption system, such as sugars, minerals, organic acids, etc., which are not adsorbed on the adsorption resin are removed, and then the olive components that have an effect on the activation of Tie2 are concentrated, and then the The one obtained by spray drying was used as an olive fruit extract. The obtained olive fruit extract was dissolved in dimethylarsin (DMSO) to a concentration of 50 μg / mL and 100 μg / mL to prepare a test sample. A test sample in which angiogenin-1 was dissolved in DMSO to a concentration of 500 ng / mL was prepared and used as a positive control group.

<Test method>

The cells were grown to a monolayer of normal human umbilical vein endothelial cells (HUVEC) 96 well plate toward the planting, it was 2.0x10 ⁴ cells /0.1mL/ wells, using low serum growth medium for vascular endothelial cells (Kurabo Co., Company-made, Humedia-EG2) training overnight. Next, HUVEC cultured overnight was replaced with 0.1 mL of vascular endothelial cell basal medium (Humedia-EB2, manufactured by Kurashiki Textile Co., Ltd.) 3 hours before cell stimulation (with test sample added), and cultured again. Thereafter, 0.1 mL of a test sample dissolved in Humedia-EB2 was added to the well, and culture was performed for 20 minutes. After incubation, the amount of phosphorylated (active) Tie2 in the cells was measured using an immunoassay group (Human Phospho-Tie2 (Y992) Immunoassay, manufactured by R & D Systems) in accordance with an experimental procedure.

In addition, phosphorylated Tie2 was also measured in the same manner as the negative control group used to dissolve DMSO in the test sample.

Thereafter, the Tie2 activation rate was calculated according to the following formula, and the phosphorylation was evaluated.

Tie2 activation rate (%) = [(Measured value of phosphorylated Tie2 when test sample is added) / (Measured value of phosphorylated Tie2 in negative control group)] × 100

<Result>

The results are shown in Table 1. As shown in Table 1, it was confirmed that the olive fruit extract had Tie2 activation.

Example 2: Tie2 activation of olive fruit extract

As a test sample, a sample containing the olive fruit extract prepared in Example 1 at a concentration of 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / mL in DMSO was used to investigate the activation of Tie2 by Western blotting. effect.

<Test method>

Cells (Ba / F3-human Tie2) showing excessive human Tie2 in mouse pro-B cells (Ba / F3) were used for Tie2 phosphorylation analysis. The stimulus of Ba / F3-human Tie2 produced by the test sample was obtained by adding RPMI-1640 medium without FBS and the test sample 10 minutes later with PBS After washing the cells, the cell extract was recovered by PhosphoSafe (trademark), Extraction Reagent (Novagen) and implemented. This was electrophoresed on a 7.5% SDS gel and transferred to a PVDF membrane. After blocking non-specific proteins with Blocking one-P (Nacalai Tesque) for 60 minutes, the band of phosphorylated Tie2 was detected using an anti-phosphorylated Tie2 antibody (Cell Signaling Technology) and an HRP-labeled secondary antibody. In addition, phosphorylated Tie2 was similarly evaluated for the dissolved DMSO used in the test sample as a negative control group, and as a positive control group, the DMSO containing angiogenin-1 at a concentration of 0.4 μg / mL in DMSO was used. The sample was similarly evaluated for phosphorylated Tie2. The detection and analysis of the band were performed using an image capturing device ChemiDoc XRS Plus (Bio-Rad Laboratories) and Image Lab Software version 2.0 (Bio-Rad Laboratories), and Tie2 activation was evaluated according to the following formula.

Tie2 activation rate (%) = [(band intensity of phosphorylated Tie2 when test sample is added) / (band intensity of phosphorylated Tie2 in negative control group)] × 100

<Result>

The results of the Western blotting method are shown in FIG. 1. Assuming that the band intensity of the negative control group is 100%, the relative value is represented by a graph. From the results of FIG. 1, it was confirmed that the olive fruit extract had Tie2 activation.

Example 3: Inhibition of vascular permeability of olive fruit extract

As a test sample, a sample containing the olive fruit extract prepared in Example 1 at a concentration of 25 μg / mL and 100 μg / mL in DMSO was used to evaluate the vascular permeability caused by vascular endothelial growth factor (VEGF). Sexual hyperinhibition.

<Test method>

Six-week-old ICR mice were purchased from Shimizu experimental materials (stocks), and they were raised in advance for more than one week. Shave the back of the mouse 3 days before the test, and use the mouse with no wound on the lower back visually. In addition, 8 administration areas were set for each mouse (4 places on the left and right across the midline of the back), and evaluation was performed with n = 4.

Mice under pentobarbital anesthesia were injected with 100 μL of 1% Ivan's blue pigment prepared with physiological saline into a vein, and 15 minutes later, an equal amount of vascular endothelial growth factor (VEGF 300ng / mL final), which is a vascular permeability factor, was mixed. ) And each test sample, take 20 μL of it and administer it to the back skin using a 30G injection needle. After 40 minutes, the skin on the back was peeled off, and the pigment leaked into the skin was observed. After taking a picture, the skin tissue was cut out and the pigment was extracted with formamidine, and the OD _{620 was} measured by an absorbance meter. In addition, an area in which neither the test sample nor VEGF was administered (a control group containing only PBS (-)) and an area where only VEGF was administered was also set for each mouse to evaluate the vascular permeability inhibition produced by the test sample. effect. The test results are based on Tukey's multiple comparison test.

<Result>

The results of the Myers analysis of each test sample are shown in FIG. 2. The horizontal axis of the graph indicates the test sample and concentration, and the vertical axis indicates the amount of Ivan's blue pigment leaked from the blood vessel. In addition, the photograph shows the leakage of pigment on the back skin at that time. In addition, the vascular permeability inhibition rate of the test sample was determined from the amount of the leaked Ivan's blue, and is shown in Table 2. As shown in Table 2, it was confirmed that the olive fruit extract had a concentration-dependent permeability-inhibiting effect from blood vessels.

Example 4: Evaluation of Tie2 activation of tyranol, tyrosol, and oleuropein from olive fruit extracts

The tyrosin hydrate, tyrosol, and oleuropein contained in the olive fruit extract were separated and purified, and the Tie2 activation of these components was evaluated.

<Isolation and purification of tyrosin hydrate, tyrosol, and oleuropein>

The olive fruit extract prepared in Example 1 was supplied to an adsorption column chromatography (Dia ion HP-20, manufactured by Mitsubishi Chemical Corporation), and then collected. Tie2 active eluted fractions were separated and purified by fractionation HPLC (ODS) to isolate tyrosin hydrate, tyrosol, and oleuropein. The three isolated compounds were identified in a manner consistent with the retention time of the HPLC standard.

<Test method>

The cells were grown to a monolayer of normal human umbilical vein endothelial cells (HUVEC) 96 well plate toward the planting, it was 2.0x10 ⁴ cells /0.1mL/ wells, using low serum growth medium for vascular endothelial cells (Kurabo Co., Company-made, Humedia-EG2) training overnight.

Next, HUVEC cultured overnight was replaced with 0.1 mL of vascular endothelial cell basal medium (Humedia-EB2, manufactured by Kurashiki Textile Co., Ltd.) 3 hours before cell stimulation (with test sample added), and cultured again. Thereafter, 0.1 mL of a test sample dissolved in Humedia-EB2 was added to the well, and culture was performed for 20 minutes. After incubation, the amount of phosphorylated (active) Tie2 in the cells was measured using an immunoassay group (Human Phospho-Tie2 (Y992) Immunoassay, manufactured by R & D Systems) in accordance with an experimental procedure.

In addition, phosphorylated Tie2 was also measured in the same manner as the negative control group used to dissolve DMSO in the test sample.

Thereafter, the Tie2 activation rate was calculated according to the following formula, and the phosphorylation was evaluated.

Tie2 activation rate (%) = [(Measured value of phosphorylated Tie2 when test sample is added) / (Measured value of phosphorylated Tie2 in negative control group)] × 100

<Result>

The results are shown in Table 3. As shown in Table 1, it was confirmed that tyramine hydrate, tyrosol, and oleuropein have Tie2 activation.

Manufacturing Example 1: Capsule

The blended amounts of olive fruit extracts and additives shown in Table 4 below are weighed, mixed until uniform and prepared into a mixture, and the resulting mixture is filled into a soft blend prepared by the blended amount shown in Table 5 according to a common method. In the capsule skin, a soft capsule is obtained.

[Industrial availability]

The Tie2 activator and the like of the present invention induce the adhesion between vascular endothelial cells and stabilize the vascular endothelial cells by inhibiting the vascular endothelial cells through the excellent Tie2 activating effect of the olive fruit extract and the like, thereby improving the vascular or lymphatic aging caused by aging And prevent it.

Claims (6)

An application of olive fruit extract used for manufacturing Tie2 activator, characterized in that the raw material of the olive fruit extract is olive fruit juice, and contains as an active ingredient selected from the group consisting of tyranol hydrate, tyrosol and oleuropein One or more ingredients in a group. The use according to claim 1, wherein the olive fruit extract is soluble in an aqueous solvent. According to the use of claim 1 or 2, wherein the olive fruit extract contains a group selected from the group consisting of polyphenols, compounds containing these in the structure, and cleaved ether ether terpenes derived from the terpene portion of these compounds. 1 or more ingredients. An application of olive fruit extract for producing a vascular permeability inhibitor, which is characterized in that the raw material of the olive fruit extract is olive fruit juice, and contains as an active ingredient a member selected from the group consisting of hydrated tyranol, tyrosol and oleuropein One or more ingredients in a group. An application for producing a vascular permeability inhibitor, containing one or more components selected from the group consisting of tyrosin hydrate, tyrosol, and oleuropein. A use for producing one or more components selected from the group consisting of tyramine hydrate, tyrosol, and oleuropein for use in producing a Tie2 activator.